Infants born at a very early stage of development commonly suffer respiratory failure because of their immature lungs, primitive respiratory drive, and vulnerability to infection. Bronchopulmonary dysplasia (BPD), a chronic lung disease in premature infants, was first characterized almost 40 years ago (Northway, N. Engl. J. Med. 276:357-368 (1967); Jobe et al., Am. J. Resp. Crit Card. Med. 163:1723-1729 (2001)). As initially described, BPD was a condition that occurred primarily in infants who were of sufficient size and maturity to survive the ravages of prolonged exposure to high oxygen and positive-pressure ventilation. These were mainly infants born between 28 and 32 weeks gestation and who weighed between 1,000 and 1,500 grams at birth. Their clinical course and lung pathology reflected the consequences of severe pulmonary oxygen toxicity and lung overexpansion. The initial pathologic descriptions of BPD noted airway injury, inflammation, interstitial fibrosis, smooth muscle cell hyperplasia, and squamous metaplasia in the distal airways. Mortality among these infants was high, and long-term ventilator-dependent respiratory failure was common among survivors (Bland, Biol. Neonate 88:181-191 (2005)).
With the advent of surfactant therapy, the widespread use of antenatal steroid therapy, and the use of advanced ventilator and nutritional therapies, the epidemiology and pathophysiology of BPD have changed considerably. Now, almost two-thirds of infants who acquire BPD weigh less than 1,000 gm and are born before 28 weeks of gestation (Martinez et al., in Chronic Lung Disease in Early Infancy, pp. 21-39; New York, Marcel Dekker (2000); Bancalori et al., Id. at pp. 41-64). In contrast to past experience, when pulmonary oxygen toxicity and lung overexpansion were considered major contributors to the development of chronic lung injury, premature infants developing BPD now are exposed to lower levels of oxygen and mechanical ventilation.
In addition, the lung pathology of these extremely immature infants with BPD differs from the “classic form” of BPD. Sometimes referred to as the “new BPD,” the disease primarily is now associated with disrupted terminal lung development. Microscopic inspection of the lungs of babies who have, died from BPD reveals a partial to complete arrest in distal lung development (Husain et al., Hum. Pathol 29:710-717 (1998)). This results in peripheral lung units of babies with BPD that have reduced alveologenesis and diminished and dysmorphic microvasculature development.
Development of the pulmonary air sacs is crucial for extrauterine survival. Lung development can be divided into five overlapping stages: early embryonic (3-7 weeks of gestation), pseudoglandular (5-17 weeks of gestation), canalicular (16-26 weeks of gestation), saccular (24-38 weeks of gestation) and alveolar (36 weeks of gestation to 2 years of postnatal age) (Burri, in Lung Growth and Development, ed. J. McDonald, pp. 1-35; New York, Marcel Dekker (1997); Kotecha, Paediatr. Respir. Rev. 1:308-13 (2000); Coalson, Sem. in Neonatology 8:73-81 (2003)). For infants born at 26 weeks gestation, the saccular and alveolar stages are not complete. During the saccular stage, the walls of the lung saccules thin, and secondary crests within the wall form. The tremendous expansion of the prospective respiratory airspaces causes the formation of saccules and a marked decrease in the interstitial tissue mass. The lung looks more and more “aerated,” although it is filled with fluid originating from the lungs and from the amniotic fluid surrounding the fetus. During the alveolar stage, alveolar formation begins by an extension and thinning of the secondary crests as they mature into the walls of alveoli. Thus, alveologenesis commences after birth for most preterm infants.
Although the mechanisms causing BPD are not completely known, indirect evidence suggests that BPD results from the effects of cytokines on the developing lung (Groneck et al., Arch. Dis. Child Fetal Neonatal. Ed. 73:F1-3 (1995); Jobe et al., Early Hum. Dev. 53:81-94 (1998); Jobe et al., Curr. Opin. Pediatr. 13:124-129 (2001)). For example, intrauterine infections have been observed to increase the levels of cytokines in the lungs of premature newborns and to be associated with an increased risk for BPD. In addition, exposure of the premature lung to oxygen and ventilator therapies has been associated with an increase in cytokine levels in pulmonary effluents and with an increased risk for developing BPD. Furthermore, it is possible that there are direct genetic causes for BPD. Because several genes are thought to be critical for terminal lung development (Bourbon et al., Pediatric Research 57:38R-46R (2005)), it is possible that abnormalities in one or more of them could directly inhibit normal lung maturation and cause BPD. Moreover, it is also possible that genetic abnormalities may indirectly increase the incidence of BPD by causing premature delivery.
Recent, limited indirect evidence suggests that TGF-β may be one of the numerous cytokines involved in inhibition of terminal lung development in BPD. For example, TGF-β has been identified in the terminal airways and pulmonary effluents of premature babies (Kotecha et al., J. Pediatr. 128:464-469 (1996); Toti et al., Pediatr. Pulmon. 24:22-28 (1997); Lecart et al., Biol. Neonate 77:217-223 (2000); Saito et al., Pediatr. Res. 55:960-965 (2004)), and the level of TGF-β1 is greatest in preterm babies who develop BPD (Kotecha, 1996) and correlates with the severity of the illness (Lecart, 2000). However, it remains unknown whether this reported increase in TGF-β levels is merely a consequence of the activity of other factors that are primarily responsible for the disease, or whether TGF-β plays a more direct role in the development of BPD. For example, besides TGF-β, approximately 20 candidate genes have been identified so far that might control terminal lung development. Of these genes, 13 have been reported to be modulated in animal models or infants with BPD (Bourbon et al., Pediatr. Res. 57:38R-46R (2005)). Using gene expression profiling techniques, Perkowski and coworkers reported that 385 genes are modulated during hyperoxic lung injury in the adult mouse (Am. J. Respir. Cell Mol. Biol. 28:682-696 (2003)).
At least two studies have examined the effect of ectopic overexpression of TGF-β1 in the developing lung. For example, Vicencio et al. reported that overexpression of biologically active TGF-β1 in the lung epithelium of newborn mice was associated with inhibition of terminal lung development (Am. J. Respir. Cell Mol. Biol. 31:650-656 (2004)), and Gauldie et al. reported that infection of newborn rat lungs with an adenovirus encoding TGF-β1 affected newborn lung structure, including producing patchy areas of fibrosis (which are not seen in the new BPD). (Am. J. Pathol. 163:2575-2584 (2003)). However, it is unclear whether the changes observed in this later model were directly related to the activity of TGF-β1, because the control lungs exposed to adenovirus (without TGF-β) exhibited marked distal airway edema, suggesting the adenovirus vector itself induced inflammation. Moreover, both studies involve forced ectopic expression of TGF-β, and therefore do not model what occurs during injury to the developing lung in premature infants with BPD. Thus, these studies are not helpful in elucidating the role of TGF-β in BPD.
At the same time, other studies have suggested that TGF-β regulates early events in lung development, including branching morphogenesis (Zhao et al., Am. J. Physiol. 273:L355-362 (1997); Zhao et al., Am. J. Physiol. 21:L412-422 (1999)). However, branching morphogenesis of the fetal lung, which affects the numbers of conducting proximal airways, is completed, prior to the saccular stage of lung development (McMurty, Am. J. Physiol. Lung Cell Mol. Physiol. 282:L341-344 (2002)). Thus, defects in branching morphogenesis are associated with diseases of early lung development, such as lung hypoplasia and congenital diaphragmatic hernia, but not of BPD, a disease of inhibition or disruption of the saccular and alveolar phases of lung development. Because abnormalities in branching morphogenesis are not observed in the lungs of infants with BPD, where the number of conducting proximal airways is normal, these studies also are not helpful in elucidating whether TGF-β plays a role in the pathogenesis of BPD.
Finally, the effect of TGF-β neutralization on lung development has been investigated in certain models of pediatric lung diseases, but not in BPD. In these cases, TGF-β activity was increased in the normal lung by alterations in the binding of TGF-β to extracellular matrix (Neptune et al., Nat. Genet. 33:407-411 (2003) and Massaro et al., Am. J. Physiol. Lung Cell Mol. Physiol. 278:L955-60 (2000)) or supplying excess levels to fetal lungs growing in culture (Zhang et al., E-PASS2006 59:5166.6 (2006)). Whether neutralization of excess TGF-β produced in the injured developing lung inhibited development of BPD was not investigated. Due to differences in etiology, pathology and disease course, these investigations in which TGF-β was neutralized have not contributed to our understanding of the role of TGF-β in BPD. See, for example, Neptune et al., Nat. Genet. 33:407-411 (2003) and Massaro et al., Am. J. Physiol. Lung Cell Mol. Physiol. 278:L955-60 (2000), which reported on the role of abnormal TGF-β signaling in mouse models of Marfan's disease; and Zhang et al., E-PASS2006 59:5166.6 (2006), which reported on the role of TGF-β in hypoxic mice that are models of congenital heart disease or of pulmonary vascular diseases associated with living in high altitudes.
In summary, while some studies have suggested that TGF-β is capable of affecting terminal lung development, others have suggested that TGF-β plays a role at a much earlier stage, during branching morphogenesis, and no studies have ever directly addressed whether TGF-β plays a causative role in modulating terminal lung development in the injured lung of a premature infant at risk of developing BPD, or whether neutralization of TGF-β would have any impact on the course of disease in such infants.
BPD is an extremely important cause of infant morbidity and mortality. Behind asthma, BPD is the most costly disease of pediatric patients. The availability of surfactant therapy and other advances in treatment have improved neonatal survival without associated reduction in rates of BPD. Therefore, there exists an important need for improved methods to treat premature infants at risk of developing BPD.